Over 1000 papers have been published utilizing the radioactive carbonized microsphere method for determining the distribution of blood flow in experimental animals. A very important application of microsphere methodolofy is the determination of flow to small tissue regions where direct flow measurement techniques cannot be used because of the small size of the vessels. The accuracy of radioactive microspheres for determining blood flow in large tissue regions has been validated by comparing microsphere-measured flow against direct measurements of either arterial inflow or total venous outflow. In small tissue regions, the microsphere method has not been validated. Published evidence now suggests that microspheres may be significantly less accurate for determining flow in small tissue regions than in large tissue regions. If this is true, the data base of a significant number of the 1000+ published investigations which have used the microsphere technique is questionable, and the results might be jeopardized. We have developed methodology which allows a quantitative investigation of the distribution of carbonized microspheres and blood flow to small tissue regions. In this grant, we propose experiments which directly compare the distribution of microspheres, volume blood flow, and leukocytes. This will be accomplished by in vivo microscopy, video-densitometric volume flow measurements, and the use of fluorescent-labeled leukocytes. By an analysis of the results, we shall come to a fundamental understanding of the conditions which adversely affect the accuracy of carbonized microspheres. This understanding should lead to marked improvements in microsphere methodology, allowing it to become the powerful quantitative method within the microvasculature that it has the potential to be.